Received for publication August 11, 2006.
Revised November 20, 2006.
Accepted for publication November 29, 2006.

The Development of a Cocktail CYP2B6, CYP2C8 & CYP3A5 Inhibition Assay and a Preliminary Assessment of Utility in a Drug Discovery Setting

Abstract

Tools for studying the roles of CYP2B6, CYP2C8 and CYP3A5 in drug metabolism have recently become available. The level of interest in these enzymes has been elevated because investigations have revealed substrate promiscuity and/or polymorphic expression. This study aimed to develop a single cocktail inhibition assay for the three enzymes and assess its utility in drug discovery. Bupropion hydroxylation, amodiaquine N-deethylation and midazolam 1'-hydroxylation were chosen as probe reactions for CYP2B6, CYP2C8 and CYP3A5 and were analysed using LC-MS-MS. Kinetic analyses were performed to establish suitable conditions for inhibition assays, which were subsequently automated. CYP2B6, CYP2C8 and CYP3A5 IC₅₀ values were determined for marketed drugs and almost two hundred AZ discovery compounds from 16 separate discovery projects. For the marketed drugs, results obtained were comparable with literature values. Data were also compared with IC₅₀ values determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. In this dataset, the majority of compounds were more potent inhibitors of CYP2C9, CYP2C19, CYP2D6 and CYP3A4 than CYP2B6, CYP2C8 or CYP3A5. The potential impact of these findings on CYP inhibition strategy is discussed.

Key words: CYP inhibition, CYP2B, CYP2C, CYP3A, drug discovery, enzyme inhibitors

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